乳杆菌代谢产物RNA组分序列分析及对其部分生物学功能的研究

发布时间：2018-06-03 06:01

本文选题：序列 + CPG-ODN　；参考：《大连医科大学》2011年硕士论文

【摘要】：目的: 1.分析乳杆菌代谢产物RNA组分的序列,明确该核酸成分的性质 2.通过RNA组分在体外对Caco-2细胞TLR-9通路及对细胞增殖影响的研究,探讨其作用机制及对肿瘤细胞的作用 3.通过RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态恢复情况的研究,探讨其对肠道机械屏障及免疫细胞的作用方法: 1.乳杆菌代谢产物RNA组分序列分析:应用Solexa上机前处理过程对样品RNA进行处理,经过加接头反转录后对连接产物进行PCR扩增,进而对PCR产物做TA克隆测序。(测序委托深圳华大基因科技有限公司进行) 2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响 (1)实验分为四组,阴性对照组、阳性对照组、RNA100ug/ml组、RNA200ug/ml组。实验组中加入不同浓度RNA组分,阳性对照组中加入CPG-ODN (2)RT-PCR法检测TLR9、NF-κBp65、IL-6 mRNA水平的变化;免疫组化法检测NF-κBp65蛋白水平的变化;ELISA法检测Caco-2细胞培养液中IL-6的变化 (3)MTT法检测RNA组分对Caco-2细胞增殖的影响 3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响 (1)SPF级Balb/c小鼠随机分为正常组,重度失衡组,自然恢复组,RNA恢复组。采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型后,以RNA组分10mg/kg/天的剂量,连续3天灌胃调节,并从灌服之日起第7天提取标本,检测指标 (2)HE染色及透射电镜观察菌群失衡小鼠通过RNA组分恢复后肠粘膜绒毛形态变化 (3)透射电镜观察菌群失衡小鼠通过RNA组分恢复后派氏结中细胞形态变化结果: 1.乳杆菌代谢产物RNA组分序列分析:所得39条序列,经Blast比对其中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等 2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响 (1)与对照组相比RNA组分组TLR9及NF-κBp65、IL-6在mRNA及蛋白水平的表达量均高于阴性对照组,100ug/mlRNA组表达量低于阳性对照组,200ug/mlRNA组表达量高于阳性对照组 (2)RNA组分对Caco-2细胞增殖有抑制作用,显示出明显的时间剂量依赖性 3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响 (1)肠粘膜绒毛形态:应用回灌RNA进行恢复后,通过HE染色发现与自然恢复组相比,肠上皮细胞层细胞排列紧密,肠绒毛排列规整,肿胀、断裂消失,绒毛顶端未见坏死脱落的肠上皮细胞,且炎性细胞浸润减少。通过电镜观察发现与自然恢复组相比,微绒毛恢复整齐,细胞间连接更加紧密 (2)派氏结中细胞形态:经过回灌RNA进行恢复后,通过电镜观察发现肠壁派氏结重度失衡组与正常组相比较,淋巴细胞之间的间隙明显变大;树突状细胞伪足变短,通过回灌RNA进行恢复后,与自然恢复组相比,淋巴细胞间隙明显缩窄,树突状细胞伪足延伸结论: 1.乳杆菌代谢产物RNA组分中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等 2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR通路具有活化作用 3.乳杆菌代谢产物RNA组分对人结肠腺癌Caco-2细胞增殖有抑制作用 4.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态具有恢复作用
[Abstract]:Objective:
1. analyze the sequence of RNA components of Lactobacillus metabolites and identify the properties of the nucleic acid components.
2. through the study of the effects of RNA components on Caco-2 cell TLR-9 pathway and cell proliferation in vitro, the mechanism and the effect on tumor cells were discussed.
3. the effects of the RNA component on intestinal microflora's intestinal mucosal villous morphology and the cell morphology recovery in the Pik junction of the intestinal microflora were discussed, and the effects on the intestinal mechanical barrier and immune cells were discussed.
Method:
Sequence analysis of RNA component of 1. Lactobacillus metabolites: the sample RNA was treated by Solexa preprocessing, PCR amplification was carried out after the joint reverse transcription, and then the TA clone of PCR products was cloned and sequenced. (sequencing of Shenzhen Huada Gene Technology Co., Ltd.)
Effects of 2. RNA components of Lactobacillus metabolites on TLR9 pathway and cell proliferation in Caco-2 cells
(1) the experiment was divided into four groups, the negative control group, the positive control group, the RNA100ug/ml group and the RNA200ug/ml group. The experimental group was added to the different concentration of RNA components, and the positive control group was added to the CPG-ODN group.
(2) the changes of TLR9, NF- kappa Bp65 and IL-6 mRNA were detected by RT-PCR method; the changes of NF- kappa Bp65 protein level were detected by immunohistochemistry; ELISA assay was used to detect IL-6 changes in Caco-2 cell culture solution.
(3) the effect of RNA components on proliferation of Caco-2 cells was detected by MTT.
Effects of Lactobacillus 3. metabolite RNA on intestinal mucosal villi morphology and cell morphology in pare nodes of intestinal flora imbalance
(1) SPF grade Balb/c mice were randomly divided into normal group, severe unbalance group, natural recovery group and RNA recovery group. After using antibiotic ceftriaxone sodium to establish the intestinal microflora imbalance model in mice, the dosage of 10mg/kg/ days in RNA group was used for gastric perfusion, and the samples were extracted from the day of seventh days, and the indexes were detected.
(2) HE staining and transmission electron microscopy were used to observe the morphological changes of intestinal villi after restoration of RNA components in mice with bacterial imbalance.
(3) transmission electron microscopy was used to observe the morphological changes of the cells in the PGE junction after the recovery of RNA components.
Result:
1. sequence analysis of Lactobacillus metabolites RNA: the 39 sequences were compared with Blast, including Lactobacillus Deshi 23S rRNA and Lactobacillus rhamnophilus tRNA-Leu.
Effects of 2. RNA components of Lactobacillus metabolites on TLR9 pathway and cell proliferation in Caco-2 cells
(1) compared with the control group, the expression of TLR9 and NF- kappa Bp65 in the RNA group was higher than that in the negative control group. The expression of 100ug/mlRNA in the 100ug/mlRNA group was lower than that in the positive control group. The expression of 200ug/mlRNA group was higher than that of the positive control group.
(2) the RNA component inhibited the proliferation of Caco-2 cells, showing a significant time dose dependence.
Effects of Lactobacillus 3. metabolite RNA on intestinal mucosal villi morphology and cell morphology in pare nodes of intestinal flora imbalance
(1) the morphology of intestinal villi: after the recovery of RNA, the cell layer cells in the intestinal epithelium were closely arranged, the intestinal villi were arranged regularly, swollen, and broken, and no necrotic intestinal epithelial cells were found at the top of the villi, and the infiltration of inflammatory cells decreased. The microvilli recovered gradually and the intercellular connections were more closely linked.
(2) the cell morphology in piret's knot: after recharge of RNA, it was found through electron microscopy that the severe imbalance between the intestinal wall pies group and the normal group compared with the normal group, the gap between the lymphocytes became significantly larger, and the dendritic cells were short of the pseudo foot. After the recovery of the recharge RNA, the lymphocytic space of the lymphocyte was narrowed and the dendritic shape was obviously narrowed. Cell pseudo foot extension
Conclusion:
1. Lactobacillus metabolite RNA contains Deshi Lactobacillus 23S rRNA and Lactobacillus rhamnophilus tRNA-Leu.
2. RNA component of Lactobacillus metabolite has activation effect on TLR pathway of Caco-2 cells.
3. Lactobacillus metabolite RNA has inhibitory effect on the proliferation of human colon adenocarcinoma Caco-2 cells.
4. the RNA components of Lactobacillus metabolites restored the morphology of intestinal mucosal villi and the morphology of cells in PASH nodes in mice with intestinal flora imbalance.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【参考文献】